Does anyone have a successful protocol for immunohistochemical staining of mouse toes? In particular, which fixation technique was used, was decalcification necessary, and were frozen sections or paraffin sections more successful? Thank you.
Dear Adriana,
Since mouse toe has bone, you certainly need decalcification. You may fix toes in 15ml 10% neutral buffered formalin solution for 48 hours in a closed tube. Rinse them in PBS for 30min two three times to get rid of fixative. The tissue can be decalcified in 10% EDTA solution (pH 7.4) for 10 days with a change in solution every 3 days.
If you need how to make EDTA, let me know: [email protected]
May be you store the tissue for long time duration period so first you can fixed in 10% formalin and then you can use any time for any histological experiment and change alternatively this solution.
It has been a while since I did this but I think you may be able to get away without any special treatment depending on the age of the animal, how much bone is involved. formalin fixation and paraffin embedding should do the trick.
If you feel that you cannot get nice sections, then please have a look at this nice paper from Ito's group: "Wnt activation in nail epithelium couples nail growth to digit regeneration" Nature. 2013 Jul 11; 499(7457): 228–232..
They used a decalicifation step and still were able to get stainings going. I had no such luck with tissue from jaws. The procedure can be harsh and proteins and their epitopes. In this paper they used "Nails were fixed in 10% Zinc buffered formalin at 4°C 2? Overnight [the meaning unclear; MMT], and washed in PBS twice. After decalcification in 22.5% formic acid/10% sodium citrate buffer at RT for 2 hr, nails were dehydrated through ethanol and xylene, embedded in paraffin, and cut into 6 μm sections. Following rehydration, paraffin-sectioned tissues were processed in hematoxylin and eosin, or Masson’s trichrome staining. For immunohistochemistry, antigen retrieval was performed by microwaving sections for 6 min on high-wattage setting in 1x TE buffer (pH. 8.0). ..."
The decalcification is relatively short. As said, I think you may be able to get away without any decalicifation. The digits are relatively soft. If you are just interested in the footpad, you need no decalcification. What exactly do you mean with "toe"; which aspect of the mouse foot are you interested in?
I am interested in harvesting toes from 9 week old C57BL/6 mice. I am most interested in the skin of the toe pad and not the bone itself, but dissection of this small amount of tissue has proven tricky. Currently, we are trying the dissected foot pad (mid plantar surface without the bone) to avoid the decalcification step. We have previously tried fixation of whole paws in 4% PFA followed by a decalcification step, but had no luck with the following IHC staining. I will try some of your suggestions. Thank you for your help!
You still could fix the entire paw, but then dissect just the foot pad. And I am pretty sure now, that I used non-decalified digits for sectioning. There is much more bone involved when you want to get to the foot pad.
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